The present invention concerns reagents for an ELISA determination of antibodies. More particularly, the present invention concerns a peptide-beta-lactamase conjugate. Even more particularly, the present invention relates to an enzyme-linked immunoassay (ELISA) applicable for the screening of sera from vaccinated individuals and from individuals who have been infected with hepatitis B virus (HBV) for antibodies specific for the pre-S region of the hepatitis B virus envelope protein.
The major protein component of the hepatitis B virus(HBV) envelope (env) and of subviral hepatitis B surface antigen particles (HBsAg) is a relatively hydrophobic, cysteine-rich sequence of 226 amino acids having a molecular weight of 25 kD. The antigenicity and immunogenicity of this protein (designated as S-protein) depends on the maintenance of disulphide bonds (G. N. Vyas, K. R. Rao and A. B. Ibrahim, "Australia Antigen (Hepatitis B Antigen): A Conformational Antigen Dependent On Disulphide Bonds", Science, 178, 130-1301, (1972); N. Sukeno, R. Shirachi and N. Ishida, "Reduction And D.K. Reoxidation of Particle Structure And Antigenicity", Journal Of Virology, 9, 182-183, 1972; G. R. Dressman, F. B. Hollinger, R. M. McCombs and J. L. Melnick, "Alteration Of Hepatitis B Antigen (HBAg) Determinants By Reduction And Alkylation", Journal of General Virology, 19, 129-134, 1973).
The open reading frame on HBV DNA coding for S protein (P. Charnay, E. Mandart, A. Hampe, F. Fitoussi, P. Tiollais, F. Galibert, "Localization Of The Viral Genome And Nucleotide Sequence Of The Gene Coding For The Major Polypeptide Of The Hepatitis B Surface Antigen (HBsAg)", Nucleic Acid Research, 1, 335-346, 1979; D. L. Peterson, I. M. Roberts and G. N. Vyas, "Partial Amino Acid Sequence Of Two Major Component Polypeptides Of Hepatitis B Surface Antigen", Proceedings Of The National Academy Of Science, U.S.A., 74, 1530-1534, 1977) has the capacity to code for a protein consisting of 389-400 amino acids (depending on the antigenic subtype of HBV). The DNA sequence corresponding to this reading frame and preceding the gene for S-protein (=S-gene) has been designated as the pre-S region (P. Tiollais, P. Charnay and G. N. Vyas, "Biology Of Hepatitis B Virus", Science, 213, 406-411, 1981).
Proteins larger than the S-protein (in its non-glycosylated (25 kD) form or glycosylated (29kD) form) have been conclusively identified in the HBV envelope and in HBsAg. Antigenic determinants specific for these proteins, but absent on S-protein, have been clearly discerned (A. Machida, S. Kishimoto, H. Ohnuma, K. Baba, Y. Ito, H. Miyamoto, G. Funatsu, K. Oda, S. Usuda, S. Togami, T. Nakamura, Y. Miyakawa and M. Mayumi, "A Polypeptide Containing 55 Amino Acid Residues Coded By The Pre-S Region Of Hepatitis B Virus-Deoxyribonucleic Acid Bears The Receptor For Polymerized Human As Well As Chimpanzee Albumins", Gastroenterology, 86, 910-918, 1984; A. R. Neurath, S. B. H. Kent and N. Strick, "Location And Chemical Synthesis Of A Pre-S Gene Coded Immunodominant Epitope of Hepatitis B Virus", Science, 224, 392-395, 1984; K. H Heermann, V. Goldmann, W. Schwartz, T. Seyffarth, H. Baumgarten and W. H. Gerlich, "Large Surface Proteins of Hepatitis B Virus Containing The Pre-S Sequence", Journal Of Virology, 52, 396-402, 1984).
Work with synthetic peptide analogs and with recombinant DNA containing portions of the pre-S region nucleotide sequence have established that the protein moieties of HBV (HBsAg) components with molecular weights greater than 29 kD correspond to the following sequences: (a) the "middle" protein representing the S-protein with 55 additional amino acids at the N-terminal and coded for by the pre-S region of the env gene (amino acid residues pre-S(120-174)) and (b) the "large" protein consisting of the "middle" protein with an additional 108-119 N-terminal amino acids (depending on the antigenic subtype; amino acid residues pre-S(1-119) or pre-S(12-119) (A. R. Neurath and S. B. H. Kent, "Antigenic Structure Of Human Hepatitis Viruses", Immunochemistry Of Viruses: The Basis For Serodiagnosis And Vaccines, 325-366, Edited by M. H. V. Van Regenmortel and A. R. Neurath, Amsterdam: Elsevier, 1985) and containing all amino acids encoded by the HBV env gene (pre-S and S regions) (A. R. Neurath, S. B. H. Kent and N. Strick, "Location And Chemical Synthesis Of A Pre-S Gene Coded Immunodominant Epitope Of Hepatitis B Virus", Science, 224, 392-395, 1984; A. R. Neurath, S. B. H. Kent, N. Strick, P. Taylor and C. E. Stevens, "Hepatitis B Virus Containing Pre-S Gene Encoded Domains", Nature (London) 315, 154-156, 1985; H. Okamoto, M. Imai, S. Usuda, B. Tanaka, K. Tachibana, S. Mishiro, A. Machida, T. Makamura, Y. Miyakama and M. Mayumi, "Hemagglutination Assay Of Polypeptide Coded By The Pre-S Region Of Hepatitis B Virus DNA With Monoclonal Antibody: Correlation Of Pre-S Polypeptide With The Receptor For Polymerized Human Serum Albumin In Serums Containing Hepatitis B Antigens", Journal Of Immunology, 134, 1212, 1984; M. -L. Michael, P. Pontisso, E. Sobczak, Y. Malpiece, R. E. Streeck and P. Tiollais, "Synthesis In Animal Cells of Hepatitis B Surface Antigen Particles Carrying A Receptor For Polymerized Human Serum Albumin", Proceedings Of The National Academy Of Sciences, U.S.A., 81, 7708-7712, 1984; D. T. Wong, N. Nath and J. J. Sninsky, "Identification of Hepatitis B Virus Polypeptides Encoded By The Entire Pre-S Open Reading Frame", Journal Of Virology, 55, 223-231, 1985; D. H. Persing, H. E. Varmus and D. Ganem, "A Frameshift Mutation In The Pre-S Region Of The Human Hepatitis B Virus Genome Allows Production Of Surface Antigen Particles But Eliminates Binding To Polymerized Albumin", Proceedings Of The National Academy Of Sciences, U.S.A., 82, 3440-3444, 1985; W. Stibbe and W. H. Gerlich, Virology, 123, 436, 1982; A. Machida, S. Kishimoto, H. Ohnuma, H. Miyamoto, K. Baba, K Oda, T. Nakamura, Y. Miyakawa and M. Mayumi, Gastroenterology, 85, 268 (1983)).
The pre-S sequences of the middle and large HBV env protein have the following properties distinct from S-protein: (1) a high proportion of charged amino acid residues and high hydrophilicity; (2) absence of cysteine residues; and (3) the highest HBV subtype-dependent amino acid sequence variability among HBV DNA gene products and minimal homology with pre-S sequences corresponding to nonhuman hepadnaviruses. These properties indicate that the pre-S sequences: (1) are exposed on the surface of HBV (HBsAg); (2) may determine the host range of HBV, in accordance with their involvement in attachment of the virus to hepatocytes; (3) have a disulphide-bond-independent antigenicity and immunogenicity confirmed experimentally (A. R. Neurath, S. B. H. Kent, N. Strick, "Location And Chemical Synthesis Of A Pre-S Gene Coded Immunodominant Epitope Of Hepatitis B Virus", Science, 224, 392-395, 1984); and (4) are recognized by the host's immune system (A. R. Neurath, S. B. H. Kent, N. Strick, "Location And Chemical Synthesis Of A Pre-S Gene Coded Immunodominant Epitope of Hepatitis B Virus", Science, 224, 392-395, 1984). Antigenic determinants located on pre-S sequences are more immunogenic than S-protein determinants located on the same HBsAg particle (D. R. Milich, G. B. Thornton, A. R. Neurath, S. B. H. Kent, M. -L. Michael, P. Tiollais and F. V. Chisari, "Enhanced Immunogenicity Of The Pre-S Region Of Hepatitis B Surface Antigen", Science, 228, 1195-1199, 1985).
The presence of pre-S sequences enhances the immune response to S-protein and circumvents the immunologic non-responsiveness to S-protein in non-responder mouse strains (Milich et al., 1985, supra; P. Coursaget, J. L. Barnes, J. P. Chiron and P. Adamowicz, "Hepatitis B Vaccines With And Without Polymerized Albumin Receptors", Lancet, 1, 1152-1153, 1985).
Antibodies with anti-pre-S specificity are virus-neutralizing. Accordingly, an immune response to pre-S sequences may play an important role in protection against HBV infection. Therefore, it is important to determine whether or not humans vaccinated against hepatitis B develop antibodies to pre-S-specific determinants. However, sufficiently sensitive assays for such antibodies have not heretofore been available.
HBV vaccines consisting of HBsAg prepared from serum (M. R. Hilleman, E. B. Buynak, W. J. McAleer, A. A. McLean, P. J. Provost and A. A. Tytell, Viral Hepatitis, W. Szmuness, H. J. Alter and J. E. Maynard, eds., The Franklin Institute Press, Philadelphia, 385, 1982) or by recombinant DNA techniques (W. J. McAleer, E. B. Buynak, R. Z. Maigetter, D. E. Wampler, W. J. Miller and M. R. Hilleman, Nature (London), 307, 178, 1984) which contain only the S-protein (A. R. Neurath, S. B. H. Kent, N. Strick, P. Taylor and C. E. Stevens, Nature (London), 315, 154, 1985) have good efficacy (C. E. Stevens, P. E. Taylor, M. J. Tong, P. T. Toy and G. N. Vyas, "Viral Hepatitis And Liver Disease", G. N. Vyas, J. L. Dienstag and J. H. Hoofnagle, eds., Grune & Stratton, Orlando, Fla., p. 275, 1984) except when administered to immunocompromised recipients (C. E. Stevens, H. J. Alter, P. E. Taylor, E. A. Zang, E. J. Harley and W. Szmuness, N. Eng.. J. Med., 311, 496, 1984; J. Desmyter and J. Colaert, Viral Hepatitis And Liver Disease, G. N. Vyas, J. L. Dienstag and J. H. Hoofnagle, eds., Grune & Stratton, Orlando, Fla., p. 709, 1984). Serum-derived HBsAg vaccines, not submitted to treatments known to destroy the pre-S gene coded portions of the HBV env proteins (A. R. Neurath, S. B. H. Strick, P. Taylor and C. E. Stevens, 1985, supra) appeared to have a better efficacy in immunocompromised individuals than did the vaccine containing S-protein only (J. Desmyter and J. Colaert, 1984, supra; J. Desmyter, J. Colaert, G. DeGroote, M. Reynders, E. E. Reerink-Brongers, P. N. Lelie, P. J. Dees and H. W. Reesink, Lancet, 2, 1323, 1983).
The following findings suggest the possibility that such differences may be explained by the absence of presence of pre-S gene coded sequences in the vaccines: (1) humans recovering from hepatitis B have antibodies to pre-S gene coded determinants on the HBV middle and large env proteins (A. R. Neurath, S. B. H. Kent, N. Strick, P. Taylor and C. E. Stevens, 1985, supra); (2) such antibodies specifically interfere with the attachment of HBV to hepatocytes (A. R. Neurath, S. B. H. Kent, N. Strick, P. Taylor and C. E. Stevens, 1985, supra); (3) at least some of these antibodies are virus-neutralizing; (4) the pre-S gene coded antigenic determinants are more immunogenic than S-protein determinants located on the same HBsAg particle ( D. R. Milich, G. B. Thornton, A. R. Neurath, S. B. H. Kent, M. -L. Michel, P. Tiollais and F. V. Chisari, Science, 228, 1195, 1985); (5) immunologic nonresponsiveness to S-protein can be overcome by immunization with pre-S gene coded sequences of the HBV env middle protein (D. R. Milich, G. B. Thornton, A. R. Neurath, S. B. H. Kent, M.-L. Michel, P. Tiollais and F. V. Chisari, 1985, supra); and (6) the presence of pre-S sequences enhances the immune response to S-protein (P. Coursaget, J. L. Barres, J. P. Chiron and P. Adamovicz, Lancet, 1, 1152, 1985), (D. R. Milich, G. B. Thornton, A. R. Neurath, S. B. H. Kent, M. -L. Michel, P. Tiollais and F. V. Chisari, 1985, supra).
In order to better understand the protective responses elicited by hepatitis B vaccines, it would be desirable to test: (1) hepatitis B vaccines for the presence and level of pre-S sequences and (2) recipients of the vaccines for the corresponding humoral and cell-mediated immune responses. Another problem of high significance and medical importance is the detection in human sera of antibodies to the lymphotropic virus type III (HTLV III/LAV). These antibodies serve as a marker for past or present infection with HTLV III/LAV. They can be a tool for screening of blood donors to assure the safety of blood and blood products. Presently available methods for detection of these antibodies are not sufficiently specific and require additional confirmatory tests.
The enzyme-linked immunoassay technique is increasingly popular because it is very sensitive and does not require specialized equipment, as do immunofluorescence and radioimmunoassay. The method depends on conjugation of an enzyme to either an antigen (Ag) or an antibody (Ab) and use of the enzyme activity as a quantitative label. Many variations of the method can be constructed, depending on the nature of the enzyme employed and the Ag-Ab system to be measured. A widely employed variant is the enzyme-linked immunosorbent assay (ELISA), which can be used to measure either Ag or Ab. To measure Ab, the known Ag is fixed to a solid phase (e.g., plastic cup or microplate), incubated with test serum dilutions, washed and then incubated with anti-immunoglobulin labeled with an enzyme (e.g., horseradish peroxidase). Enzyme activity is measured by adding the specific substrate: the color reaction is estimated colorimetrically. The enzyme activity is a direct function of the amount of antibody bound.
To measure Ag, a known specific Ab is fixed to the solid phase, the test material containing Ag is added and washed, and a second enzyme-labeled Ab is added. This test requires that the Ag have at least two determinants. After washing, substrate is added and enzyme activity is estimated colorimetrically and related to Ag concentration.
U.S. Pat. Nos. b 3,654,090 and 4,343,896, describe methods for determining antigens and antibodies using antigens or antibodies covalently linked to enzymes. Enzymes described therein include peroxidase, beta-glucuronidase, beta-D-glucosidase, beta-D-galactosidase, urease, glucose oxidase+peroxidase and galactose oxidase+peroxidase.
Enzymes considered to be suitable for ELISA are described in U.S. Pat. No. 3,839,153. Such enzymes listed include catalase, peroxidase, beta-glucuronidase, beta-D-glucosidase, beta-D-galactosidase, urease, glucose oxidase, galactose oxidase and alkaline phosphatase.
An enzymatic immunological method for the determination of antigens and antibodies is described in U.S. Pat. No. 4,106,043. The preferred enzyme in U.S. Pat. No. 4,106,043 is horseradish peroxidase. Other listed enzymes include catalase, peroxidase, urease, glucose oxidase and alkaline phosphatase.
U.S. Pat, Nos. 4,169,012 and 4,228,240 are directed to a stabilized peroxidase reagent for enzyme immunoassay, i.e., peroxidase and a metal ion, e.g., Mg, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Co, Zn, Ga and Al.
U.S. Pat. No. 3,850,752 lists the following enzymes for coupling to a hapten, protein or antibody: catalase, peroxidase, beta-glucuronidase, beta-D-glucosidase, beta-D-galactosidase, urease, glucoseoxidase and galactose-oxidase.
U.S Pat. No 3,879,262 concerns the detection and determination of haptens. The following enzymes are describe for use therein: catalases, peroxidases, glucuronidases, glucosidases, galactosidases, urease and oxidoreductases (glucose oxidase and galactose oxidase).
Although a number of different hepatitis B vaccines have good efficacy in immunocompetent recipients, there are obvious differences between distinct vaccines (Desmyter and Colaert, 1984 supra). The variety of hepatitis B vaccines is expected to increase in the near future. Therefore, the need for studies comparing their immunogenicities using standardized techniques will also increase. These should include methods for detection and quantitation of all HBV-specific proteins in the vaccines and the quantitation of specific antibodies elicited in vaccine recipients.